Six distinct fractions that exhibited binding activity were isolated from rabbit antiserum against substance P (SP) by pH gradient elution on a CM-Sephadex column chromatography. Isoelectric points (IP) and SP-binding characteristics were determined on some of these fractions. Two of them had IP of 9.0 while another one had a value of 8.0. One of the major binding fractions appeared to have binding sites with two different affinity for SP while a minor fraction with a single binding site. Partially purified antiidiotypic antibodies were prepared and shown to compete with SP for binding to SP receptors in brain and salivary gland. The IC-50 for cold SP and antiidiotype serum for these two tissues were 7.5 and 1.4 mM and 1:1320 and 1:600 dilution, respectively. In a separate study, the membrane protein from cerebral cortex was solubilized and purified with wheat germ lectin Sepharose chromatography. Affinity gel was prepared by coupling the solubilized membrane protein on Affi-Gel 10 and its binding activity and the effect of Mn++ were measured. This is the first report confirming that the SP binding activity of the solubilized membrane protein is intact. Finally, the relationship between the development of SP receptor and the neuropeptide itself was studied. High affinity specific binding sites for SP appeared to develop late in the gestation period and this development is largely in parallel with the development of substance P content of the tissue.